Carotenoids are known as natural pigments with colors ranging from yellow through orange to red or purple, widely found in nature. Astaxanthin, one of carotenoids, is widely used in feeds for enhancing the color of fish and chicken eggs. Astaxanthin is also approved as a food additive and widely used in fat processed foods, protein foods, aqueous liquid foods, health foods, and the like. Moreover, since astaxanthin has strong antioxidant effects such as antioxidative activity against free radical-induced lipid peroxidation, and singlet oxygen scavenging activity which is several hundred times more powerful than that of α-tocopherol, nowadays astaxanthin is expected to be used in cosmetics, functional foods and drugs so that its antioxidant effects may prevent the generation of excessive active oxygen in the body, for example, to improve skin blemishes and wrinkles; to prevent cataract, arteriosclerosis, and heart disease; to enhance immunity; and to prevent cancer.
Astaxanthin is widely found in nature, for example, in fishes such as salmons, trouts, and red sea breams; and crustaceans such as crabs, shrimps, and krills. Also, astaxanthin is produced by microorganisms such as bacteria belonging to the genus Paracoccus, the genus Brevibacterium, and the like; algae such as Hematococcus pluvialis; and yeasts such as Xanthophyllomyces dendrorhous (also known as Phaffia rhodozyma). Carotenoids such as astaxanthin and zeaxanthin currently widely used are provided by chemical synthesis in terms of stable supply and costs. However, chemically synthesized carotenoids have the risk of contamination of process-related contaminants and particularly poisons that are used in synthesis reactions and harmful to humans. Thus, supplying naturally-derived products is desired in terms of safety.
With such a background, many reports have been published to disclose methods for producing carotenoids including astaxanthin using natural algae and microorganisms that are considered to be suitable for industrial mass production. For example, a method using Haematococcus algae is reported in which cultured cyst cells of algae are treated with hot acetone to elute chlorophyll that is a contaminant, and then astaxanthin is extracted with ethanol (Patent Literature 1). The carotenoid composition obtained by this method, however, contains many biological contaminants. Thus, this method has difficulty in industrially producing a carotenoid composition with a sufficient amount of astaxanthin. In addition, it is difficult for this method to remove the acetone used in the production to reduce it to, for example, a level equal to or less than the amount specified in food additive regulations.
Another report (Patent Literature 2) discloses a method for producing a composition containing 0.5 to 60% of astaxanthin without using organic solvents harmful to humans. This method includes kneading Haematococcus algae with broken cell walls with a cosolvent such as water or glycerin to give a formed body, filling an extraction tank with the formed body, and supplying supercritical fluid thereto to extract astaxanthin. However, for example, mass production by this method needs special equipment and also requires a long period of time for extraction to achieve a sufficient yield. Besides, the composition obtained by this method needs additional concentration for achieving a high astaxanthin content. Thus, this method cannot satisfy simplicity and cost efficiency for industrial production of a highly-concentrated carotenoid composition.
A method using a yeast of the genus Xanthophyllomyces (the genus Phaffia) is also reported in which the yeast cells are subjected to extraction in an n-hexane-ethanol mixed solvent while they are crushed with a compression crusher; the extract is concentrated to give an oily substance; the oily substance cooled at −50° C. is suspended and then filtered to remove free fatty acids and triglycerides; and residual free fatty acids in the pigment oil are further removed through conversion to metal soaps, whereby a high concentration of astaxanthin can be obtained (Patent Literature 3). Still another report (Patent Literature 4) discloses a method including extracting disrupted cells of the genus Xanthophyllomyces (the genus Phaffia) with an organic solvent, concentrating the extract to give an oily crude extract, and refining the crude extract by a refining method such as ion-exchange chromatography or adsorption chromatography to obtain astaxanthin or an ester thereof. These methods, however, include complicated processes. In particular, the latter method includes a troublesome step of refining a low-concentration crude astaxanthin solution by multiple column chromatography operations. Thus, industrial production by these methods is difficult.
Meanwhile, other reports (Patent Literatures 5 and 6) disclose preparation of dry products containing 40% or more of astaxanthin by extracting a novel bacterium (E-396 strain) capable of producing astaxanthin, with a lower alcohol or a mixture of a lower alcohol and water, concentrating the extract to give a precipitate, and washing the precipitate with lower alcohols. These methods enable preparation of a highly-concentrated astaxanthin composition only using water and a highly safe solvent such as ethanol. In the method of Patent Literature 5 including extraction at 80° C. or higher, however, the extraction needs to be performed in a sealable pressure vessel and such a high-temperature operation may convert part of naturally-derived free trans-astaxanthin to the cis form, which unfortunately may reduce the refining yield of astaxanthin having the desired structure. Moreover, the methods of Patent Literatures 5 and 6 are unclear if they are applicable to species other than the novel bacterium that has been actually confirmed to enable the preparation of such astaxanthin compositions.
Still another report (Patent Literature 7) discloses a production method that includes extracting cultured yeast cells of the genus Xanthophyllomyces (the genus Phaffia) with acetone, concentrating the extract to give a crude extract, and adding a hydrocarbon solvent to the crude extract to cause crystallization. This method has some advantages such as high simplicity and capability of obtaining a composition with a high astaxanthin content of 40% or higher. However, this method, which uses a hydrocarbon solvent in the production, may allow this solvent to remain in the resulting composition.
In particular, yeasts of the genus Xanthophyllomyces are known to contain about 25% by weight of lipids per dry cell weight, and extracts from these yeasts mainly contain lipids. Thus, concentrating such an extract results in a sticky, viscous concentrate. For this reason, when a composition with a high concentration of astaxanthin is prepared using a yeast of the genus Xanthophyllomyces, many kinds of contaminants such as lipids need to be removed during the preparation process. Accordingly, this requires a complicated and non-industrial process or an organic solvent such as hexane which is harmful to humans and the residual amount of which in final products should meet regulations.
In such circumstances, desired is a simple method for producing a naturally-derived carotenoid composition having a high astaxanthin content but a low residual solvent content or a residual solvent content that causes no problem.